Name: preparation, characteristics, toxicity, and efficacy evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo
Uploaded: 2022-09-28T13:40:00
Description: Watch this Scientific Journal Video about Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo at JoVE.com

本研究由国家自然科学基金项目项目第31670938号、32070924号32000651、重庆市自然科学基金项目编号2014jcyjA0107、2019jcyjA-msxmx0159、陆军军医大学专项项目编号2020XBK24、2020XBK26号、国家大学生创新创业计划202090031021号、第202090031035号支持。

动物实验按照《实验动物—动物福利伦理评审导则》（GB/T 35892-2018）进行，并经第三军医大学实验动物福利与伦理委员会批准。通过腹膜内注射100mg / kg的1%戊巴比妥钠对小鼠实施安乐死。
1. I-OVA NE的准备
<ol><li>将1mg单磷酸脂质A（MPLA）与100μLDMSO混合，涡旋5分钟，并在室温（RT）下静置4小时以完全溶解。</li>
	<li>定量加入吐温80和I-OVA并混合。 注：吐温80和I-OVA?...

<ol>
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用免疫细胞膜功能化的纳米疫苗在疾病靶向治疗中具有很大的优势，并且通过独特的肿瘤嗜性、特异性靶点的识别、延长的循环、增强的细胞间相互作用和低全身毒性等特性将副作用降至最低。它们还可以轻松地与其他治疗模块集成，以合作治疗癌症16，20。通过控制物理和化学性质（如测量、形状和电荷）可以获得所需的属性。因此，纳米疫苗在广泛?...

作为最重要的公共卫生创新之一，疫苗在抗击全球人类疾病负担方面发挥着关键作用1。例如，目前正在测试120多种COVID-19疾病的候选疫苗，其中一些已在许多国家获得批准2。最近的报告指出，癌症疫苗有效地改善了临床癌症治疗的进展，因为它们指导癌症患者的免疫系统识别出身体外来的抗原3。此外，位于肿瘤细胞内部或外部的多个T细胞表位可用于设计肽疫苗，由于缺乏与放疗和化疗相关的显着毒性，其在治疗转移性癌症方面显示出优势4，5。自1990年代中期以来，主要使用抗原肽疫苗进行肿瘤治疗的临床前和临床试验，但很少有疫苗对癌症患者表现出足够的治疗效果6。此外，具有肽表位的癌症疫苗免疫原性差，递送效率不足，这可能是由于从给药部位迅速扩散的细胞外肽快速降解，导致免疫细胞抗原摄取不足7。因此，有必要用疫苗输送技术克服这些障碍。
OVA 257-264是MHC I类结合257-264表位，表达为融合蛋白，是一种常用的模型表位8。此外，OVA257-264 对针对肿瘤的适应性免疫应答至关重要，这取决于细胞毒性T淋巴细胞（CTL）反应。它由肿瘤中的抗原特异性CD8 + T细胞介导，这些CD8 + T细胞由OVA257-264 肽诱导。其特征是颗粒酶B不足，由细胞毒性T细胞释放，导致靶细胞凋亡8。然而，游离的OVA257-264 肽给药可能诱导很少的CTL活性，因为这些抗原的摄取发生在非特异性细胞而不是抗原呈递细胞（APC）中。缺乏适当的免疫刺激导致CTL活性5。因此，有效的CTL活性的诱导需要相当大的进步。
由于上皮细胞提供的屏障和粘液的持续分泌，疫苗抗原迅速从鼻粘液中去除9，10。开发一种可以穿过粘膜组织的高效疫苗载体至关重要，因为抗原呈递细胞位于粘膜上皮9下方。鼻内注射疫苗理论上诱导粘膜免疫以对抗粘膜感染11。此外，鼻腔给药是一种有效且安全的疫苗给药方法，因为它方便，避免肠道给药，并改善了患者的依从性7。因此，鼻腔给药是新型肽表位纳米疫苗的良好给药手段。
已经设计了几种合成生物材料来结合细胞 - 组织和细胞 - 细胞相互作用的表位。某些生物活性蛋白质，如Ile-Lys-Val-Ala-Val（IKVAV），已被引入作为水凝胶结构的一部分，以赋予生物活性12。这种肽可能有助于细胞附着、迁移和生长13 ，并结合整合素α3β1 和α 6β1 与不同的癌细胞类型相互作用。IKVAV是一种细胞粘附肽，来源于层粘连蛋白基底膜蛋白α 1 链，最初用于模拟神经微环境并引起神经元分化14。因此，为这种新型疫苗找到有效的输送载体对于疾病控制非常重要。
最近报道的乳液系统，如W805EC和MF59，也已用于灭活流感疫苗或重组乙型肝炎表面抗原的鼻腔递送，并说明可触发粘膜和全身免疫15。与颗粒粘膜递送系统相比，纳米乳液（NEs）具有易于给药和与有效佐剂共形成方便的优点16。据报道，纳米乳剂疫苗以不同于传统脱敏的持续方式改变过敏表型，从而导致长期抑制作用17。其他人报告说，纳米乳剂与Mtb特异性免疫显性抗原相结合可以诱导有效的粘膜细胞反应并赋予显着的保护18。因此，设计了一种以合成肽IKVAV-OVA 257-264（I-OVA，由与OVA257-264结合的IKVAV组成的肽）的新型鼻内自组装纳米疫苗。系统地评估这种新型纳米疫苗非常重要。
该方案的目的是系统地评估纳米疫苗的理化特性、毒性和稳定性，检测抗原摄取以及是否利用技术手段增强保护和治疗效果，并阐述主要实验内容。在这项研究中，我们建立了一系列方案来研究其理化特性和稳定性，确定CCK-8对BEAS-2B细胞的I-OVA NE的毒性大小，并使用共聚焦显微镜观察BEAS-2B细胞对疫苗的抗原呈递能力，评估这种新型纳米疫苗在体内和体外的释放曲线。，并使用E.G7-OVA荷瘤小鼠模型检测该疫苗的保护和治疗效果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates</td>
			<td>Corning Incorporated, USA</td>
			<td>CLS3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad 6.0 microplate reader</td>
			<td>Bio-Rad Laboratories Incorporated Limited Co., CA, USA</td>
			<td>&#160;Bio-Rad 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 kits</td>
			<td>Dojindo, Japan</td>
			<td>CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf, Germany&#160;</td>
			<td>5811000398</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Hyclone (Life Technology, USA)</td>
			<td>SH30088.03</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-labeled I-OVA</td>
			<td>Shanghai Botai 
			Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HF 90/240 Incubator</td>
			<td>Heal Force, Switzerland</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC&#160;</td>
			<td>Shanghai Botai Biotechology Co., Ltd.</td>
			<td>E2695</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon,Japan</td>
			<td>DSZ5000X</td>
			<td></td>
		</tr>
		<tr>
			<td>IPC-208</td>
			<td>Chong Qing University, China</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS system&#160;</td>
			<td>Caliper Life Science Limited Company</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>JEM-1230 TEM</td>
			<td>JEOL Limited Company of Japan</td>
			<td>1230 TEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern NANO ZS</td>
			<td>Malvern Instruments Ltd., UK</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MPLA</td>
			<td>&#160;Invivogen 
			Lit. Co.</td>
			<td>tlrl-mpla</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin Sulfate Ointment</td>
			<td>Shanghai CP General Pharmaceutical Co. , Ltd.</td>
			<td>H31022262</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA257&#8211;264</td>
			<td>Shanghai Botai 
			Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Hyclone (Life Technology, USA)</td>
			<td>SH30809.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic peptide (I-OVA) conjugation of IKVAV-PA</td>
			<td>Shanghai Botai 
			Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM800 laser scanning confocal fluorescence microscope</td>
			<td>Zeiss, Germany</td>
			<td>Zeiss LSM800</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation, characteristics, toxicity, and efficacy evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo

表位肽因其安全性、高特异性、生产方便等特点在肿瘤疫苗领域受到广泛关注;特别是，一些MHC I限制性表位可以诱导有效的细胞毒性T淋巴细胞活性以清除肿瘤细胞。此外，鼻腔给药是一种有效且安全的肿瘤疫苗递送技术，因为它具有便利性和提高患者依从性。然而，表位肽因其免疫原性差且缺乏递送效率而不适合鼻腔递送。纳米乳液（NEs）是热力学稳定的系统，可以加载抗原并直接递送到鼻粘膜表面。Ile-Lys-Val-Ala-Val（IKVAV）是层粘连蛋白的核心五肽，层粘连蛋白是一种由人呼吸道上皮细胞表达的整合素结合肽。本研究采用低能量乳化法制备了含有合成肽IKVAV-OVA257-264（I-OVA ）的鼻内自组装表位肽NE肿瘤疫苗。IKVAV和OVA257-264 的组合可以增强鼻粘膜上皮细胞的抗原摄取。在这里，我们建立了一个协议，通过透射电子显微镜（TEM），原子力显微镜（AFM）和动态光散射（DLS）研究物理化学特性;粘蛋白存在下的稳定性;通过检查BEAS-2B细胞的细胞活力以及C57BL / 6小鼠的鼻和肺组织的毒性;通过共聚焦激光扫描显微镜（CLSM）摄取细胞;通过 体内小动物成像释放曲线;以及通过使用E.G7荷瘤模型的疫苗的保护和治疗效果。我们预计该协议将为新型T细胞表位肽粘膜疫苗的未来开发提供技术和理论线索。

根据该方案，我们完成了鼻肿瘤纳米疫苗递送的制备和体外和体内实验评估。TEM、AFM 和 DLS 是评估纳米疫苗表面 zeta 电位和粒径的基本特性的有效手段（图 1）。BEAS-2B上皮细胞是鼻腔疫苗体外毒性测试的有用筛选模型（图2A）。用H&E染色的显微照片表明I-OVA NE没有明显的粘膜毒性，包括组织损伤，出血或炎症细胞浸润（<strong class="xfi...

Watch this Scientific Journal Video about Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo at JoVE.com

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

在这里，我们提出了鼻自组装纳米乳肿瘤疫苗体 外 和体内制备和评价的详细方法 。

鼻自组装纳米乳肿瘤疫苗体外和体内的制备、特性、毒性及疗效评价

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

新型纳米乳疫苗在疾病策略治疗中具有很大的优势。这些集合效应通过独特的肿瘤切碎、特定策略的鉴定和低全身毒性等特性而最小化。我们预计该方案将为新型T细胞ectype肽粘膜疫苗的未来开发提供技术和理论线索。首先将一毫克单磷脂质A与100微升DMSO混合。涡旋五分钟，在室温下溶解四小时。定量添加吐温80和I-OVA。混合成分并加入角鲨烯。接下来，向制备的混合物中加入100微升MPLA溶液。为了制备纳米乳液疫苗，将混合溶液以大约占总体积的70%加入水滴中，轻轻搅拌以获得透明且易于流动的混合物。对于体外测定，从恢复BEAS2B上皮细胞开始。打开水浴并将温度调节到37摄氏度。在水浴中快速解冻冷冻细胞瓶，在37摄氏度下回火。解冻后，将细胞快速移液到15毫升无菌离心管中，并加入两毫升完整的生长培养基。将试管以 129 G 离心五分钟。除去舒嘎吱并将细胞重悬于两毫升完全生长培养基中。再次离心细胞后，除去脱敏剂并加入六毫升完全生长培养基以重悬细胞。然后将细胞转移到T25培养瓶中，并在5%二氧化碳培养箱中以37摄氏度孵育。一旦细胞密度达到80%至90%，丢弃培养基并用两毫升PBS洗涤细胞两次。加入一毫升0.25%tripsin以消化细胞一到两分钟。当观察到细胞变圆时，立即加入四毫升完整的生长培养基以中和胰蛋白酶。在15毫升无菌离心管中混合并吸出样品。将样品以 129 G 离心五分钟。取出舒替，将细胞重悬于一毫升RPMI 1640完全培养基中。使用20微升细胞悬液进行细胞计数，并将细胞稀释至每毫升10至第五个细胞的1倍。然后将BEAS2B细胞以每孔10倍至第四个细胞的密度接种在100微升RPMI 1640完全培养基中的96孔板中，并将板预孵育24小时如前所述。在孵育结束时，丢弃嘀嘀嗒，并向96孔板的每个孔中加入100微升RPMI 1640。加入I-OVA纳米乳液各100微升，I-OVA与BNE混合，I-OVA用各种终浓度的完全生长培养基预稀释，BNE作为对照，并在37摄氏度下孵育24小时。孵育完成后，取出培养基，然后将板转移到黑暗的地方。向板的每个孔中加入90微升完全生长培养基和10微升CCK8溶液。将板在37摄氏度和5%二氧化碳下孵育两小时。使用酶标记的酶标仪在450纳米处测量每个孔的吸光度。使用10微升移液器吸头对小鼠进行I-OVA，I-OVA加BNE和I-OVA NE的鼻免疫三天。使用BNE和PBA作为实验对照。之后，用剪刀切割约三毫米厚的鼻组织样本，并将它们放入4%多聚甲醛中。取出肺组织，将鼻和整个肺组织固定在4%多聚甲醛中24小时。TEM和AFM用于评估纳米疫苗表面zeta电位和粒径的基本特性。显示了I-OVA NE在穆辛稳定性分析中的粒径、多分散指数、zeta电位和电泳迁移率。BEAS2B 细胞的相对活力在暴露于不同肽浓度的 I-OVA、BNE+I-OVA 和 I-OVA NE 24 小时的培养物中超过 80%。I-OVA EN组无明显的黏膜毒性、组织损伤、出血或炎症细胞浸润。上皮BEAS2B细胞捕获的FITC标记的I-OVA NE比其水溶液更多。采用IV-IS系统分析I-OVA NE体外缓释效果。与I-OVA的水溶液相比，I-OVA NE疫苗具有缓释作用。在I-OVA NE组中，存活时间百分比高于其他组，肿瘤体积明显较小，表明其比其他组具有更好的预防保护效果。在治疗模型中，不同组之间的中位生存时间显着不同。I-OVA NE组的肿瘤体积明显小于I-OVA组。在这个过程中，均匀搅拌混合物很重要。否则会降低纳米疫苗的效率。

Research

JoVE Journal

Immunology and Infection

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...